Tooling up for safer genome editing

A genome editing tool uses an enzyme to make changes in DNA sequences more specific without the risk of damage that accompanies the use of the CRISPR-Cas9 system

The CRISPR-Cas9 system opened a new area in genome engineering, enabling researchers to modify almost any genome in the evolutionary tree leading up to humans with unprecedented ease and the possibility to swap disease-rendering mutations in the DNA for ‘healthy’ sequences. This widely pursued approach, however, is still suffering from the fact that current CRISPR-Cas9-based technologies run the risk of damaging the genome instead of repairing it.

This is because the Cas9 enzyme first has to break the double-stranded DNA helix before it can fix it. After cutting the target DNA, Cas9 engages one of two natural repair mechanisms, called homology-directed repair (HDR), to enter a desired change from a ‘healthy’ template strand into the targeted DNA. To the chagrin of many researchers, HDR is often outcompeted by the second repair mechanism, non-homologous end joining (NHEJ), that also recognizes the cut but introduces unwanted mutations and rearrangements into the DNA that defeat the purpose of repair.

Programmable cytidine deaminases use the ability of cytidine deaminase enzymes to change a single atom on cytidine — one of the four standard bases that constitute the universal genetic code — to convert it into thymidine. As a consequence, C-G basepairs are substituted by T-A basepairs in the DNA doublehelix to re-write the genetic code. By fusing the enzyme to specific DNA recognition proteins called TALEs or zink fingers, the enzyme’s activity can be directed toward specific DNA sequences within the genome to edit DNA without the need for breaking it. Credit: ESB Professional/Shutterstock

To help circumvent these problems in current genome editing efforts, George Church’s research team at the Wyss Institute and Harvard Medical School (HMS) has developed an alternative tool to edit DNA. The method harnesses the ability of a family of enzymes called cytidine deaminases to convert cytidine (C) — one of the four base units that together make up the genetic code — to another one, thymidine (T). Astonishingly, it accomplishes this transition by exchanging merely a single atom of nitrogen for oxygen, without breaking the DNA strands.

The researchers fused the cytidine deaminase alternatively to two DNA binding modules known as zinc fingers and transcription activator-like effectors (TALEs), that are engineered to directly latch on to specific DNA sequences. This way they directed the enzyme’s activity to cytidine bases in the immediate vicinity, creating a fully programmable base editor.

“Our customized cytidine deaminase fusions have several potential advantages over CRISPR editing: they don’t need a co-delivered template strand and work independently of guide RNAs. They are smaller than CRISPR-Cas9, which could facilitate viral delivery to target cells and tissues” said Church, Ph.D., is a Core Faculty Member at the Wyss Institute for Biologically Inspired Engineering at Harvard University and Professor of Genetics at Harvard Medical School. “Since they don’t unwind or cut the DNA double helix, like Cas9 does, this could enable several changes at once with less toxicity to cells.”

Church’s team showed that the method effectively converts Cs to Ts in the genome of bacteria and human cells, while avoiding the kind of genome vandalizing chromosomal abnormalities that can occur with CRISPR-Cas9 use. However, because the engineered deaminase enzymes are still displaying some unspecific activity in the genome, the researchers are now working to increase their specificity also by taking pages from the playbook of CRISPR-Cas9 development.

In theory, more than 300 human diseases could benefit from a conversion of C to T alone, but in the future, this approach could be even broadened for example by co-opting members of another family of deaminases that convert adenines (A) to guanines (G). Since the genetic code is anchored by C-G and A-T base pairing between the two strands of the DNA double helix, this means that all of the four standard bases could one day be specifically mutated by deaminase targeting.

The cytidine deaminase-based genome editing tool developed by Church’s team was presented in a recent article in Nature Communications.